Our ongoing clinical trials of immunotherapy for pediatric sarcomas are targeting the tumor specific fusion proteins generated by the t(11;22) and t(2;13) in Ewing's sarcoma and alveolar rhabdomyosarcoma respectively. Thus far, we have noted difficulty in generating measurable immune responses toward these targets. In an attempt to potentially identify new targets for immunotherapy and to investigate whether endogenous immune responses might already exist in patients with Ewing's sarcoma, we have conducted a series of experiments to study anti-tumor CTL toward Ewing's sarcoma. Autologous T cells were collected by apheresis from patients with Ewing's sarcoma and were tested for reactivity toward autologous tumors. These studies showed that cytolytic T cells are already primed in vivo and circulate in patients with Ewing's sarcoma. These tumor reactive killers can be expanded and activated by CD40L-matured DCs following by re-stimulation with tumor cells, or anti-CD3 and 4-1BB antibodies in the absence of autologous tumor. Using such methods, we generated tumor-lytic T cell responses in 4/4 patients. However, polyclonal stimulation using anti-CD3/anti-CD28 beads, followed by tumor cell stimulation did not induce similar tumor-lytic responses. Phenotypic analysis showed that responding cells in these cultures were CD8+/CD28-/4-1BB (CD137)+. Interestingly, they were oligoclonal, resided in the memory T cell pool and expressed NK receptors, ( KIR and NKG2D) which mediated non-MHC class I restricted lysis of allogeneic tumor targets. 4-1BB receptor is a member of the tumor necrosis factor receptor (TNFR) superfamily that is preferentially expressed on the surface of CD8+ T cells after antigen-induced activation. Cross-linking of 4-1BB and T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Thus, during the course of these studies we investigated whether Ewing's sarcoma itself might be capable of providing the 4-1BB mediated costimulatory signal. We discovered that Ewing's sarcoma cell lines themselves express 4-1BB ligand. Furthermore, T cell activation could be induced by the tumor alone but required both signal one (presumably provided by a tumor associated antigen) and signal two which is provided by 4-1BB ligand expressed by the tumor. When either of these interactions are blocked, the tumor induced proliferation of autologous T cells is blocked. These results provide new evidence that endogenous immune responses exist in patients with Ewing's sarcoma and question the widely held view that progressive tumor growth induces T cell tolerance. Furthermore, these results open the intriguing possibility that tumor expression of an immune mediated co-stimulatory molecule may provide ongoing signals which sustain an immune response in vivo. We are currently developing approaches to expand these tumor reactive killers and to study whether they are capable of controlling tumor growth in an animal model. We further plan to investigate whether similar tumor:T cells 4-1BBL:4-1BB interactions are relevant in other tumor models. These results are currently being submitted for publication [Tumor Expression of 4-1BB Ligand: Sustenance for Tumor Specific Cytolytic T Cells? Hua Zhang, Kevin S Chua, Chand Khanna Lee J Helman, Bill Telford, Yvona Ward, Elaine K Thomas and Crystal L Mackall, submitted.] These studies were carried out in part through a CRADA developed with Immunex Corp. to obtain CD40 ligand for dendritic cell differentiation as well as various reagents for blocking 4-1BB/4-1BBL interactions.